Comparative Study On The Regulation Of Gene Expression Levels By Combinations Of Eukaryotic Expression Regulatory Elements

As binding sites of transcription factors,cis-acting elements regulate the precise initiation and transcription efficiency of gene transcription.Currently,the function of most individual cis-acting elements has been thoroughly analyzed in mammalian cells.To detect the regulation of gene expression by different combinations of cis-acting elements,this study constructed expression vectors containing different combinations of regulatory elements and used fluorescence,quantitative real-time PCR(q RT-PCR),and western blotting to detect the expression of the downstream reporter gene e GFP regulated by different combinations of cis-acting elements.The effects of four promoters(PGK promoter,Polr2 a promoter,and EF-1α core promoter),two enhancers(CMV enhancer and SV40enhancer),two introns(EF-1α intron A and hybrid intron),and two terminators(CYC1 terminator and TEF terminator),as well as their different combinations,on downstream gene expression were compared in various mammalian cells.To verify the effects of these combinations of regulatory elements in practical applications,this experiment also used the receptor binding domain(RBD)sequence of the novel coronavirus spike protein to replace the e GFP sequence in the expression vector and detected the regulatory effects of these regulatory elements and their combinations on RBD expression in HEK-293 T cells by q RT-PCR and western blotting.This study mainly compared the effects of different combinations of regulatory elements on downstream gene transcription and translation levels.The results showed that the EF-1α core promoter had higher promoter activity,the CMV enhancer had stronger enhancer activity,the EF-1α intron A had higher intron activity,and the TEF terminator had stronger terminator activity.It was also found that gene expression levels could be regulated by optimizing the combination of cis-acting elements.The results showed that the expression of the e GFP protein in the expression vector containing the combination of CMV enhancer,EF-1α core promoter,and TEF terminator was approximately three times higher than that of the original vector in different animal cells,and the expression of the recombinant RBD protein in HEK-293 T cells was approximately 2.63 times higher than that of the original vector.In addition,this study found that the combination of multiple regulatory elements that could upregulate gene expression did not necessarily exhibit a synergistic effect to further enhance expression.These findings contribute to the optimization of expression vectors used in synthetic biology and related fields and can be applied in biological applications requiring the regulation of gene expression.